A quick, cheap, and reliable protocol for immunofluorescence of pluripotent and differentiating mouse embryonic stem cells in 2D and 3D colonies

Summary Immunofluorescent labeling is a widely used method to visualize endogenous proteins. It can be expensive and difficult to stain mouse embryonic stem cells (mESCs) because they require expensive growth media, prefer specific substrates, grow in 3D, and have loose cell-substrate adhesion. Here we propose a half-a-day, cheap, easy-to-follow, and reproducible protocol for immunofluorescence of mESCs. This protocol has been streamlined to allow a fast visualization of the investigated proteins, and we provide tips specific to stem cell culture. For complete details on the use and execution of this protocol, please refer to Chaigne et al. (2021).1

SUMMARY Immunofluorescent labeling is a widely used method to visualize endogenous proteins. It can be expensive and difficult to stain mouse embryonic stem cells (mESCs) because they require expensive growth media, prefer specific substrates, grow in 3D, and have loose cell-substrate adhesion. Here we propose a half-a-day, cheap, easy-to-follow, and reproducible protocol for immunofluorescence of mESCs. This protocol has been streamlined to allow a fast visualization of the investigated proteins, and we provide tips specific to stem cell culture. For complete details on the use and execution of this protocol, please refer to Chaigne et al. (2021). 1

BEFORE YOU BEGIN
The goal of this protocol is to stain endogenous proteins with a cheap and fast method while preserving the 3D structure of colonies of cells. This protocol is also suitable for 3D organoid cultures embedded in Matrigel. The main advantage of this method is that is uses very small wells allowing the use of small amounts of media and antibodies which reduces the cost, and preserves the 3D architecture.
The immunofluorescence protocol below describes the specific steps using mouse embryonic stem cells (mESC). However, this protocol can also be used for other mammalian cell cultures. Before you start, you need to prepare the specific cell media required for the experiment. Naïve mouse embryonic stem cells are grown in 2i/LIF conditions (containing two inhibitors of the Erk1/2 signaling pathway and of Glycogen Synthase Kinase 3 (GSK3) and Leukemia Inhibitory Factor (LIF). Cells exiting naïve pluripotency are cultured in N2B27. The culture media conditions for mouse embryonic stem cells have been developed in the Smith lab. 2 Mouse embryonic stem cells can be plated on two different substrates. If you choose to plate them on gelatin, follow option 1. The main advantage of plating cells on gelatin is that it maintains the 3D organization of mESC. It does, however, make them harder to image. On the other hand, option 2 offers plating cells on laminin where they grow in 2D, allowing faster imaging and easier visualization of proteins. Here we present a protocol for cultures that have been growing during 48 h, but numbers can in principle be scaled linearly for shorter culture (24 h, 36 h) or longer culture (72 h, 96 h). Culture longer than 96 h is not recommended.
We will discuss two variations of the protocol: the first one is a standard protocol which works for most of the proteins (here we propose to stain for a-Tubulin and actin using Phalloidin), and the second is a variant of that protocol which is specified for cortical proteins like NuMA (here we propose to stain for a-Tubulin and NuMA).
Culture of 3D colony of mouse embryonic stem cells Note: The wells need to be covered with gelatin for at least 15 min at room temperature (20 C-25 C) prior to seeding the cells. These plates have small wells which enable less antibody usage per sample. Bigger plates, e.g., 24-well or 96-well plate can also be used for higher throughput.

Plating of the cells
The following steps are the same for both options.
6. Remove media from the cells and add prewarmed Accutaseä. 7. Leave it on the cells for 3 min in the incubator at 37 C and at 7% CO 2 . Accutase TM can be left a few more min on the cells if need be.
Note: 1 ml of Accutaseä is enough for a 10 cm 2 cell culture dish. Accutaseä needs to be stored at À20 C long term or 4 C short term. Keeping it at room temperature for longer time will inactivate the enzyme.
8. Carefully resuspend the cells and pipette up and down to obtain a single cell solution.
Note: Check under the microscope if the cells are not in clumps before proceeding to the next step. If cells are still in clumps, leave the Accutaseä for a few extra min, not exceeding 10.

OPEN ACCESS
10. Centrifuge the cells for 3 min at 1000 rpm/1500 g. 11. Remove the media containing Accutaseä and leave the pellet of cells. 12. Add 1 mL of DMEM F12 + 5% BSA and resuspend the cells. 13. Remove the coating from the wells which you have previously coated. 14. Add enough media ($200 mL per well) to cover the bottom of the wells. 15. Plate the cells at the required concentration in the wells.
Note: When using 8-well plates with area of 2.20 cm 2 , plate 30000 cells for a 48 h culture.
16. After 48 h cells are ready to be stained.
Note: Cells need to be attached to the bottom of the wells before you proceed with the protocol.
17. Before starting the staining, prepare all the solutions required. That includes fixative-permeabilization and blocking buffer. Details of the solutions can be found in the materials and equipment section.

MATERIALS AND EQUIPMENT Preparation of N2
Note: N2 can be stored for 2 years at À80 C.

Components of 2i/LIF media
Note: 2i/LIF can be stored for 2 weeks at 4 C.

PHEM buffer
Note: Adjust pH at 6.9. This solution can be stored for 6 months at RT.
CRITICAL: Work with formaldehyde and TCA under the fume hood.
Alternatives: Paraformaldehyde can also be used instead of formaldehyde.

STEP-BY-STEP METHOD DETAILS Fixation and permeabilization
Timing: 10-30 min Ideally, fixation should immobilize targeted antigens, but not disturb the cell structure. 3 Here, we propose 2 options. Our main protocol proposes to fix and permeabilize at the same time for gain of time. Our second option uses TCA and proposes to fix then permeabilize. Most antibodies work well with simultaneous formaldehyde fixation/permeabilization.
Note: If the simultaneous protocol does not work well, try to separate fixation and permeabilization. If this does not work well, try with a different fixative (0.2% glutaraldehyde, ice cold TCA or ice cold methanol).
1. Delicately remove the media from the well using a pipette at the corner of the well.
Note: Remove the media and at the same time add the fixation/permeabilization solution (130 mL per well).

Incubate the cells in the solution for 10 min at room temperature (20 C-25 C).
Note: To preserve better osmolarity, fixative and permeabilization solution can be prepared in PHEM buffer. Prepare fresh fixation/permeabilization solution each time you are doing the experiment.
Alternative: For the 2-steps protocol use 10% ice cold TCA as a fixative. Fix cells for 20 min at 4 C.

Wash the fixative by rinsing the cells with PBS one time.
Alternative: For the 2-steps protocol rinse the cells with DPBS three times for 5 min. Permeabilize the cells by incubating them in 0.5% Triton TM -X for 5 min. Rinse with DPBS (optional).
CRITICAL: This step should be done as fast as possible to prevent tissue or cell deformations. However, all rinses should be done carefully so the cells are not washed away from the well. Pause Point: If needed, it is possible to pause after washing the fixative for up to 10 days. In that case, always rinse the cells three times for 5 min with PBS, wrap with stretch film to seal and store at 4 C.

Timing: 15 min
The blocking step helps minimizing any unspecific antibody binding within the cell.

Timing: 2 h
To visualize the proteins of interest, use a specific antibody. You can use multiple primary antibodies in the same experiment. If using multiple primary antibodies, make sure that they originate from different species to avoid cross-reactivity when using secondary antibodies to visualize the proteins. 5. Prepare the antibody solution by diluting the primary antibodies in the blocking solution.
Note: As a starting point, we find that most of the antibodies give a good result at a 1:200 ratio (1 mL of antibody can be diluted in 200 mL of the blocking solution). However, you can follow the instructions given for a specific antibody.
6. Incubate 130 mL of primary antibody solution for 2 h at room temperature (20 C-25 C) on a shaker at 20 rpm.
Note: No humid chamber is needed. If needed, the primary antibodies can be incubated overnight (16 h) at 4 C.

Secondary antibody
Timing: 1 h The secondary antibodies should be specific to the species of the primary antibodies and have conjugated fluorophores in the wavelengths of interest. You can mix the secondary antibodies. Note: As a starting point, we find that most of the antibodies give a good result at a 1:500 ratio (1 mL of antibody can be diluted in 500 mL of the blocking solution). However, you can follow the instructions given for a specific antibody.
10. Incubate with 130 mL of the secondary antibody solution for 1 h at room temperature (20 C-25 C) on a shaker at 20 rpm.
Alternative: Phalloidin can be used to label actin at a 1:200 ratio and can be mixed with secondary antibodies.
Note: Depending on the microscope used for visualization, you can use multiple different secondary antibodies. This depends on the laser separation of your microscope. We recommend using a simple confocal microscope with a good spectral separation.

DNA labeling
Timing: 10 min If needed, the DNA can be stained as the final step of this protocol using Hoechst (1:10000) or DAPI (0.1 mg/mL).

EXPECTED OUTCOMES
Following this protocol should allow the quick and cheap visualization of a broad range of proteins. From top to finish the protocol can be reduced to $4 h. Using this protocol, mouse embryonic stem cells are cultured in small wells. This allows smaller media consumption, as well as using less antibodies for staining and allowing high-throughput testing of antibodies. Skipping the mounting step and imaging the cells directly in a well allow to preserve the 3D structure of colonies.

LIMITATIONS
The usual limitations of immunofluorescence protocols apply. In particular, this protocol is only suitable for fixed cells, and some proteins are not well labeled by commercial antibodies. Autofluorescence, linked to the cell type, the fixation or blocking solutions, can interfere with fluorescence coming from the fluorochromes on secondary antibodies. A clear limitation of this specific protocol is the timing of imaging. Since cells are imaged directly in the culture well in PBS, the sample needs to be visualized as soon as possible to avoid possible bacterial contaminations.

TROUBLESHOOTING Problem 1
When plating 3D culture of mouse embryonic stem cells, colonies seeded on gelatin can be easily washed from the well or coverslip during the washing steps.

Potential solution
To improve adhesion, before coating the wells with gelatin, plasma activate the wells for 30s, using a plasma cleaner at maximal intensity. Plasma cleaning uses activated and ionized gas to break any impurities present on the substrate and allows for better adhesion of the coating reagent and the cells.

Potential solution
Try different fixation protocols, e.g., ice cold methanol or 4% paraformaldehyde. The PHEM buffer increases the quality of staining of cytoskeletal protein. Ice cold methanol can be used to visualize cross-linking proteins such as PRC1, as formaldehyde and paraformaldehyde are cross-linking fixation agents which disrupt PRC1 structure. A quenching step can be added after fixation and permeabilization (15 min in 1 mg/mL À1 Sodium Borohydride in CMF-PBS (Ca and Mg free PBS)).

Problem 3
Proteins or molecules of interest cannot be nicely visualized because.
The background staining is too high; The intensity of the target protein is too low.

Potential solution
Increase the concentration of Triton-X TM to increase the permeabilization. Change the blocking buffer (BSA or milk) and the antibody buffer. Increase the concentration of BSA or milk. Increase the concentration of secondary and/or primary antibody.

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate/analyze datasets/code.